The present invention relates to the sterile sampling of a liquid and, more particularly, to the periodic sterile withdrawal from a liquid flow path of a sample quantity of the liquid. In its most preferred respects, the invention relates to a method and apparatus for periodically effecting the sterile withdrawal of a sample of culture fluid utilized for the in vitro culture of animal cells.
In many systems for the in vitro culture of animal cells either for recovery of cell-secreted proteins therefrom or for other purposes such as growth of tissue-like masses or study of cellular interactions or the like, culture medium which is used to nourish the cells and to carry away cell-secreted products is continuously or intermittently fed to and withdrawn from the culture chamber through appropriate tubing, oftentimes in a recirculating closed loop system with provision for periodic or continuous replenishment of fresh culture medium.
It is well known that the monitoring of the culture medium in the culture unit and/or at one or more points in the flow path is a useful way for monitoring and controlling the culture process. Thus, for example, analysis of the medium before and after passage through the culture chamber for nutrient components and/or cell-secreted proteins and/or cell-secreted metabolites or the like can provide important information regarding the number of viable cells in the culture unit, the rates of nutrient consumption by the cells and the rate of product secretion, the cell growth rates, the particular stages of cell growth or subdivision, and the like, all of which can be used not only to monitor the system but to signal changes which might require alteration of process conditions, medium composition, or the like designed to optimize the culturation process.
Also well-known is the fact that the culturing process must be carried out under the strictest of aseptic conditions lest the cells, which are without the elaborate defense mechanisms generally provided by a typical in vivo animal environment, become contaminated, leading to contamination of products recovered therefrom and/or loss of cell viability. As a consequence, in vitro animal cell culture systems and their component parts are initiated and maintained under sterile conditions, with each portion or the entirety of the system being sterilized prior to commencement of the process, and using sterile culture medium and uncontaminated seed cell stocks.
The problem arises, then, of the need to insure that sampling of the culture medium or culture fluid be carried out in a manner which avoids introduction of contaminants into the pre-established sterile system. Prior known techniques for accomplishing this sterile withdrawal of liquid for sampling are, at best, elaborate, time-consuming and expensive, and at worst, ineffective. Generally, the area from which a liquid sample is to be taken, be it a culture vessel or a liquid flow line to or from a culture vessel, is provided with a sample port such as in the form of a short segment of tubing or other appropriate structure. The system is then invaded via this sample port to withdraw a quantity of liquid. Some techniques involve the use of an elastomeric septum in the sample port into which a sterilized needle, affixed to a syringe, is inserted and withdrawn. Such techniques are recognized as being far from ideal and run significant risk of introducing contaminants into the culture system via the sample port, particularly where the same sample port is used for repeated sampling. More sophisticated sampling techniques involve connection of a sample bottle or like container to the sampling port to permit a liquid sample to collect in the bottle, followed by removal of the bottle from the port for analysis of the collected liquid contents, all being effected through a series of complex valves or other suitable devices associated with the sample port and/or sample bottle. Still other techniques involve utilization of sterilizable connectors between the sample port and collection receptacle, wherein the connection is sterilized before and/or after the collection receptacle is affixed and removed, e.g., by flushing of the connector with a sterilizing gas.
As a consequence either of the inefficiency or complexity of known sampling techniques, not only is the risk of contamination present but there also may exist an inherent limitation on the number or frequency of samplings which can be accommodated, either by reason of a limited number of sterilizable sequences to which a particular connector can be subjected before severe degradation occurs or simply by reason of the inordinate amount of time needed to perform a sample withdrawal. This can pose significant problems in situations where rapid and frequent sampling is required in order to monitor a potentially fast-changing situation. Still further, of course, elaborate and/or time-consuming sampling techniques can add significantly to the overall cost of the culture process.
It is accordingly an object of the present invention to provide a method and apparatus for effecting the withdrawal of a liquid sample from a sterile liquid container or flow line, which permits of rapid and cost-effective performance of the sampling operation yet under conditions whereby the sterile environment is safeguarded. More specific objects of the invention are to provide a method and apparatus for sterile withdrawal of a sample of culture fluid from an on-going in vitro culturation of animal cells, be it from the culture chamber, flow lines leading to or from the culture chamber, or from other apparatus or flow lines associated with the culture system.